Patent application title: AraC in combination with a cytokine-secreting cell and methods of use thereof

Abstract:

The present invention provides improved method of cancer therapy in a
mammal. More particularly, the invention is concerned with systems
comprising cytosine arabinoside (AraC) and a cytokine-expressing cancer
immunotherapy composition and methods of administering the combination to
cancer patients in order to generate an immune response against the
cancer and provide treatment with therapeutic efficacy that is an
improvement relative to administration of AraC or the cytokine-expressing
cancer immunotherapy composition alone as a monotherapy.

Claims:

1. An improved method of cancer therapy, the improvement comprising:
administering cytosine arabinoside (AraC) and a cytokine-expressing
cancer immunotherapy composition to a subject with cancer, wherein the
administration results in enhanced therapeutic efficacy relative to
administration of the cytokine-expressing cancer immunotherapy
composition or the AraC alone.

3. The method of claim 2, wherein the cells of said cytokine-expressing
cancer immunotherapy composition are autologous to the subject.

4. The method of claim 2, wherein the cells of said cytokine-expressing
cancer immunotherapy composition are allogeneic to the subject.

5. The method of claim 2, wherein the cells of said cytokine-expressing
cancer immunotherapy composition are bystander cells.

6. The method of claim 2, wherein the cells of said cytokine-expressing
cancer immunotherapy composition are rendered proliferation-incompetent
by irradiation.

7. The method of claim 2, wherein the subject is a mammal.

8. The method of claim 7, wherein the mammalian subject is a human.

9. The method of claim 2, wherein the cancer is selected from the group
consisting of acute myeloid leukemia, prostate cancer, non-small cell
lung carcinoma and pancreatic cancer.

10. The method of claim 9, wherein the cancer is an acute myeloid
leukemia.

11. The method of claim 4, wherein the allogeneic cells are a
cancer-derived cell line, the cell line selected from the group
consisting of an acute myeloid leukemia line, a prostate cancer line, a
non-small cell lung carcinoma line and a pancreatic cancer line, wherein
the cell line is derived from the same type of cancer as the cancer of
the subject.

12. The method of claim 11, wherein the allogeneic cells are an acute
myeloid leukemia-derived cell line, wherein the cancer of the subject is
acute myeloid leukemia.

15. The method of claim 1, wherein the AraC is administered
subcutaneously.

16. The method of claim 1, wherein the AraC is administered
intraperitoneally.

17. The method of claim 1, wherein the AraC is administered intravenously.

18. The method of claim 1, wherein the AraC is administered intrathecally.

19. The method of claim 1, wherein administration of the combination
results in a long-lasting tumor-specific immune response against the
cancer.

20. The method of claim 2, further comprising administration of an
additional cancer therapeutic agent or treatment.

21. The method of claim 20, wherein the additional cancer therapeutic
agent is expressed by a cell and the cell is an autologous, allogeneic or
a bystander cell.

22. The method of claim 21, wherein the autologous, allogeneic or a
bystander cell is rendered proliferation-incompetent by irradiation.

23. The method of claim 1, wherein the AraC is administered prior to, at
the same time as, or following the administration of the
cytokine-expressing cancer immunotherapy composition.

24. The method of claim 23, wherein the AraC is administered prior to the
administration of the cytokine-expressing cancer immunotherapy
composition.

25. An improved system for cancer therapy, comprising;a combination of
AraC and a cytokine-expressing cancer immunotherapy composition, wherein
the combination is co-administered to a subject with cancer, wherein said
co-administration results in enhanced therapeutic efficacy relative to
administration of the c cytokine-expressing cancer immunotherapy
composition or AraC alone.

27. The system of claim 26, wherein the cells of said cytokine-expressing
cancer immunotherapy composition are autologous to the subject.

28. The system of claim 26, wherein the cells of said cytokine-expressing
cancer immunotherapy composition are allogeneic to the subject.

29. The system of claim 26, wherein the cells of said cytokine-expressing
cancer immunotherapy composition are bystander cells.

30. The system of claim 26, wherein the cells of said cytokine-expressing
cancer immunotherapy composition are rendered proliferation-incompetent
by irradiation.

31. The system of claim 28, wherein the allogeneic cells are a tumor cell
line selected from the group consisting of an acute myeloid leukemia, a
prostate tumor line, a non-small cell lung carcinoma line and a
pancreatic cancer line, wherein the cell line is derived from the same
type of cancer as the cancer to be treated.

32. The system of claim 31, wherein the allogeneic cells are an acute
myeloid leukemia-derived cell line, wherein the cancer to be treated is
acute myeloid leukemia.

33. The system of claim 25, wherein AraC is administered by the
intraperitoneal, subcutaneous, intravenous or intrathecal route.

34. The system of claim 25, wherein the cytokine-expressing cancer
immunotherapy composition is administered subcutaneously, intratumorally,
or intradermally.

35. The method of claim 25, wherein the AraC is administered prior to, at
the same time as, or following the administration of the
cytokine-expressing cancer immunotherapy composition.

36. The system of claim 34, wherein the AraC is administered prior to the
administration of the cytokine-expressing cancer immunotherapy
composition.

37. The system of claim 25, wherein administration of said combination
provides a long-lasting tumor-specific immune response against the
cancer.

[0002]The present invention relates to compositions and methods of
preventing and/or treating cancer in a mammal. More particularly, the
invention is directed to compositions and methods comprising a
combination of cytosine arabinoside (AraC) and a cytokine-secreting cell
and methods of administering the combination in order to treat cancer and
generate a specific, long term immune response to cancer cells in a
patient.

BACKGROUND OF THE INVENTION

[0003]The immune system plays a critical role in the pathogenesis of a
wide variety of cancers. When cancers progress, it is widely believed
that the immune system either fails to respond sufficiently or fails to
respond appropriately, allowing cancer cells to grow. Currently, standard
medical treatments for cancer including chemotherapy, surgery, radiation
therapy and cellular therapy have clear limitations with regard to both
efficacy and toxicity. To date, these approaches have met with varying
degrees of success dependent upon the type of cancer, general health of
the patient, stage of disease at the time of diagnosis, etc. Improved
strategies that combine specific manipulation of the immune response to
cancer in combination with standard medical treatments may provide a
means for enhanced efficacy and decreased toxicity.

[0004]The use of cancer cells as vaccines to augment anti-cancer immunity
has been explored for some time (Oettgen et al., "The History of Cancer
Immunotherapy," In: Biologic Therapy of Cancer, Devita et al. (eds.) J.
Lippincot Co., pp. 87-199, 1991). However, due to the weak immunogenicity
of many cancer cells, e.g., down regulation of MHC molecules, the lack of
adequate costimulatory molecule expression and secretion of
immunoinhibitory cytokines by cancer cells, the response to such vaccines
has not resulted in long term efficacy. See, e.g., Armstrong T D and
Jaffee E M, Surg Oncol Clin N Am. 11(3):681-96, 2002 and Bodey B et al.,
Anticancer Res 20(4):2665-76, 2000.

[0005]Numerous cytokines have been shown to play a role in regulating the
immune response to tumors. For example, U.S. Pat. No. 5,098,702 describes
using combinations of Tumor Necrosis Factor (TNF), Interleukin-2 (IL-2)
and Interferon-β (IFN-β) in synergistically effective amounts
to combat existing tumors. U.S. Pat. Nos. 5,078,996, 5,637,483 and
5,904,920 describe the use of Granulocyte macrophage colony-stimulating
factor (GM-CSF) for treatment of tumors. However, direct administration
of cytokines for cancer therapy may not be practical, as they are often
toxic when administered systemically. (See, for example, Asher et al., J.
Immunol. 146:3227-3234, 1991 and Havell et al., J. Exp. Med.
167:1067-1085, 1988.)

[0009]Acute myeloid leukemias (AMLs) are highly malignant neoplasms
responsible for a large number of cancer-related deaths. AML is a cancer
of the myeloid line of white blood cells, characterized by the rapid
proliferation of abnormal cells which accumulate in the bone marrow and
interfere with the production of normal blood cells. The American Cancer
Society estimates that 11,930 individuals in the U.S. will be diagnosed
with AML annually. AML is quite resistant to currently available
treatments, and approximately 76% of these patients will die of their
disease (Deschler and Lubbert Cancer 2006; 107(9):2099-107; Jemal et al.
Cancer statistics, 2006;56(2): 106-30). The 5-year survival rates range
from 36% in patients younger than 45 years to only 1.3% in patients older
than 75 years (Lowenberg et al., The New England journal of medicine
1999;341(14):1051-62; Kern and Estey Cancer 2006;107(1):116-24).
Remission induction therapies using cytosine arabinoside (AraC), a
nucleotide analogue (at a dosage of, for example, 100 to 200
mg/m2/day for 5 to 10 days) induce complete remissions in ˜75%
of younger adults and -50% in patients who are older than 60 (Jabbour et
al., Mayo Clinic proceedings 2006;81 (2):247-60; Kayaga et al Gene
therapy 1999;6(8):1475-81). However, without intensive post-remission
therapies, greater than 95% of all AML patients are destined to relapse
(Abou-Jawde et al. Leukemia & lymphoma 2006;47[4]:689-95; Stone, Seminars
in hematology 2002;39[3 Suppl 2]:4-10).

[0010]Post-remission therapy options include repeated cycles of high-dose
AraC (e.g., 10 injections of 3000 mg/m2 AraC) or high dose
myelo-ablative chemotherapy combined with either autologous or allogeneic
stem cell transplant. Whereas dose intensification of AraC during
consolidation has been shown to be associated with lower relapse rates in
younger patients, treatment options for patients over the age of 60 are
limited. Most of these patients do not tolerate high dose AraC-based
consolidation chemotherapy regimens well. The dose-limiting toxicities of
AraC are severe neutropenia and lymphopenia, which are associated with
higher mortality rates (Stone et al. The New England journal of medicine
1995;332(25): 1671-7). Therefore, the anti-leukemic benefit of increasing
the dose of AraC is offset by increased treatment-related mortality.
Another post-remission therapeutic option is hematopoietic stem cell
transplantion, but this is a difficult procedure, and is also not a good
option for older patients due to excessive treatment-related mortality.

[0011]Given the limitations of AraC treatment, and that the use of
genetically modified cancer cells as anti-cancer vaccines has met with
success in treatment of some forms of cancer, there remains a need for
improved treatment regimens with greater potency/efficacy and less side
effects than the therapies currently in use.

SUMMARY OF THE INVENTION

[0012]The invention provides compositions and methods for the treatment of
cancer in a mammal, typically a human, by administering a combination
comprising a cytokine-expressing cellular vaccine and cytosine
arabinoside (AraC).

[0014]In another aspect of the invention, the cytokine-expressing cellular
vaccine is rendered proliferation-incompetent by irradiation.

[0015]In yet a further aspect of the invention, administration of the
combination results in enhanced therapeutic efficacy relative to
administration of the cytokine-expressing cellular vaccine or AraC alone.

[0016]In yet another aspect of the invention, the cytokine-expressing
cellular vaccine is administered subcutaneously, intratumorally, or
intradermally.

[0017]In yet another aspect of the invention, AraC is administered
intravenously.

[0018]In another aspect of the invention, AraC may be administered prior
to, at the same time as, or following the administration of the
cytokine-expressing cellular vaccine component of the combination.

[0019]In yet another aspect of the invention, AraC is administered prior
to the administration of the cytokine-expressing cellular vaccine.

[0020]The invention further provides a combination, wherein the
combination comprises cells that are autologous, allogeneic, or bystander
cells.

[0021]In another aspect of the invention, the autologous, allogeneic, or
bystander cell is rendered proliferation-incompetent by irradiation.

[0022]The invention further provides compositions, methods and kits
comprising cytokine-expressing cellular vaccines in combination with AraC
for use according to the description provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIGS. 1A and 1B are schematic depictions that show tracking of tumor
burden in a mouse acute myeloid leukemia tumor model using a Xenogen IVIS
(FIG. 1A); with progress of the tumor monitored by photon counts starting
on Day 7 post challenge (FIG. 1B).

[0026]FIG. 4 is a graphic depiction of the results of a study directed to
evaluating the tumor-specific memory response following administration of
the combination of AraC and GM-CSF-secreting tumor cell in the mouse
acute myeloid leukemia tumor model. after receiving the combination
therapy, animals that survived the initial C1498.luc tumor challenge was
rechallenged with a second, larger dose of the C1498.luc cells. The
results are presented as percent tumor-free mice versus days post
rechallenge.

[0027]FIGS. 5A-5F are graphic depictions of the results of an evaluation
of the effect of treatment with HBSS, C1498.GM and AraC plus C1498.GM,
respectively in a mouse acute myeloid leukemia tumor model, indicating
51Cr release assay of splenocytes cocultured with inactivated
C1498.GM using C1498 as target cells (FIGS. 5A-C). The percentage of
purified splenocytes positive for CDI07a (FIG. 5D), CD44hi/CD62L1o (FIG.
5E), and NKG2D (FIG. 5F), in the CD8 subpopulation, as determined by flow
cytometry, is also shown.

DETAILED DESCRIPTION OF THE INVENTION

[0028]The present invention represents improved compositions for the
treatment of cancer. The compositions comprise cytosine arabinoside
(AraC) and a cytokine-secreting cellular vaccine. The invention includes
methods of administering the combination in order to enhance the immune
response to tumor cells in a patient.

[0029]The invention is not limited to the specific compositions and
methodology described herein. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to limit the scope of the present
invention.

Definitions

[0030]The terms "immune response" as used herein refers to any alteration
in a cell of the immune system or any alteration in the activity of a
cell involved in the immune response. Such alteration includes an
increase or decrease in the number of various cell types, an increase or
decrease in the activity of these cells, or any other changes which can
occur within the immune system. Cells involved in the immune response
include, but are not limited to, T lymphocytes, B lymphocytes, natural
killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells
and neutrophils. In some cases, the immune response is stimulated or
enhanced and in other cases the immune response is suppressed.
Stimulation of the immune system may include memory responses and/or
future protection against subsequent antigen challenge.

[0031]The terms "cytosine arabinoside," "Cytarabine," "AraC" and the like
as used herein refer to a nucleotide analog used as a cancer
chemotherapeutic agent. That is, a chemical agent that is toxic to,
induces apoptosis in, or blocks cell division of a cancer cell. AraC is a
standard treatment traditionally used in the treatment of cancer, e.g.,
radiation. AraC is typically administered in the form of a chemical
entity, provided in a pharmaceutically acceptable excipient. In a further
aspect, AraC is an agent or treatment, when administered to a patient in
combination with a cytokine-expressing cellular vaccine results in an
improved therapeutic outcome for the patient under treatment.

[0032]The term "cytokine" or "cytokines" as used herein refers to the
general class of biological molecules which effect/affect cells of the
immune system. The definition is meant to include, but is not limited to,
those biological molecules that act locally or may circulate in the
blood, and which, when used in the compositions or methods of the present
invention serve to regulate or modulate an individual's immune response
to cancer. Exemplary cytokines for use in practicing the invention
include but are not limited to IFN-alpha, IFN-beta, and IFN-gamma,
interleukins (e.g., IL-1 to IL-29, in particular, IL-2, IL-7, IL-12,
IL-15 and IL-18), tumor necrosis factors (e.g., TNF-alpha and TNF-beta),
erythropoietin (EPO), MIP3a, ICAM, macrophage colony stimulating factor
(M-CSF), granulocyte colony stimulating factor (G-CSF) and granulocyte
macrophage colony stimulating factor (GM-CSF).

[0033]The term "cytokine-expressing cellular vaccine" as used herein
refers to a composition comprising a population of cells that has been
genetically modified to express a cytokine, e.g., GM-CSF, and that is
administered to a patient as part of a cancer treatment regimen. The
cells of such a "cytokine-expressing cellular vaccine" comprise a
cytokine-encoding DNA sequence operably linked to expression and control
elements such that the cytokine is expressed by the cells. The cells of
the "cytokine-expressing cellular vaccine" are typically tumor cells and
may be autologous or allogeneic to the patient undergoing treatment and
or may be "bystander cells" that are mixed with tumor cells, typically
taken from the patient.

[0034]The term "operably linked" as used herein relative to a recombinant
DNA construct or vector means nucleotide components of the recombinant
DNA construct or vector are directly linked to one another for operative
control of a selected coding sequence. Generally, "operably linked" DNA
sequences are contiguous, and, in the case of a secretory leader,
contiguous and in reading frame, however, some sequences, e.g., enhancers
do not have to be contiguous.

[0035]As used herein, the term "gene" or "coding sequence" means the
nucleic acid sequence which is transcribed (DNA) and translated (mRNA)
into a polypeptide in vitro or in vivo when operably linked to
appropriate regulatory sequences. A "gene" typically comprises the coding
sequence plus any non-coding sequences associated with the gene (e.g.,
regulatory sequences) and hence mayor may not include regions preceding
and following the coding region, e.g., 5' untranslated (5'UTR) or
"leader" sequences and 3' UTR or "trailer" sequences, as well as
intervening sequences (introns) between individual coding segments
(exons). In contrast, a "coding sequence" does not include non-coding
DNA.

[0036]The terms "gene-modified" and "genetically-modified" are used herein
with reference to a cell or population of cells wherein a nucleic acid
chain has been introduced into the cell or population of cells. The
nucleic acid sequence may be heterologous to the cell(s), or it may be an
additional copy or improved version (e.g., mutated) of a nucleic acid
sequence already present in the cell(s). The cell(s) may be genetically
modified by physical or chemical methods or by the use of recombinant
viruses. Chemical and physical, and viral methods can be utilized.
Several recombinant viral vectors which find utility in effective
delivery of genes into mammalian cells include, for example, retroviral
vectors, adenovirus vectors, adenovirus-associated vectors (AAV), herpes
virus vectors, pox virus vectors. Non-viral means of introduction
include, for example, naked DNA delivered via liposomes,
receptor-mediated delivery, calcium phosphate transfection,
electroporation, particle bombardment (gene gun), or pressure-mediated
delivery may also be employed to introduce a nucleic acid chain into a
cell or population of cells to render them "gene-modified" or
"genetically-modified".

[0037]As used herein, the terms "tumor", "neoplasm" and "cancer" refer to
a cell that exhibits a loss of growth control and forms unusually large
clones of cells. Tumor, neoplasmic, or cancer cells may also have lost
contact inhibition and may be invasive and/or have the ability to
metastasize.

[0038]The term "antigen from a tumor cell" and "tumor antigen" and "tumor
cell antigen" may be used interchangeably herein and refer to any
protein, carbohydrate or other component derived from or expressed by a
tumor cell which is capable of eliciting an immune response. The
definition is meant to include, but is not limited to, whole tumor cells
that express all of the tumor-associated antigens, tumor cell fragments,
plasma membranes taken from a tumor cell, proteins purified from the cell
surface or membrane of a tumor cell, or unique carbohydrate moieties
associated with the cell surface of a tumor cell. The definition also
includes those antigens from the surface of the cell which require
special treatment of the cells to access.

[0039]As described herein, a "tumor cell line" comprises cells that were
initially derived from a tumor. Such cells typically are immortalised
(i.e., genetically modified to exhibit indefinite growth in culture).

[0040]The term "systemic immune response" as used herein means an immune
response which is not localized, but affects the individual as a whole.

[0041]The term "gene therapy" as used herein means the treatment or
prevention of a disease or medical condition, including cancer, by means
of ex vivo or in vivo delivery, through viral or non-viral vectors, of
compositions containing a recombinant genetic material.

[0042]The terms "inactivated cells," "non-dividing cells" and
"non-replicating cells" may be used interchangeably herein and refer to
cells that have been treated rendering them proliferation incompetent,
e.g., by irradiation. Such treatment results in cells that are unable to
undergo mitosis, but retain the capability to express proteins such as
cytokines or other cancer therapeutic agents. Typically a minimum dose of
about 3500 rads is sufficient, although doses up to about 30,000 rads are
acceptable. Effective doses include, but are not limited to 5000 to 10000
rads. Numerous methods of inactivating cells, such as treatment with
Mitomycin C, are known in the art. Any method of inactivation which
renders cells incapable of cell division, but allows the cells to retain
the ability to express proteins is included within the scope of the
present invention.

[0043]As used herein "treatment" of an individual or a cell is any type of
intervention used in an attempt to alter the natural course of the
individual or cell. Treatment includes, but is not limited to,
administration of e.g., a cytokine-expressing cellular vaccine, or a
cytokine-expressing cellular vaccine and at least one additional cancer
therapeutic agent or treatment, and may be performed either
prophylactically or subsequent to diagnosis as part of a primary or
follow-up therapeutic regimen. Treatment is any type of intervention that
can result in improved therapeutic outcome, which can include, but is not
limited to, induction of cancer remission, reduction of cancer relapse,
reduction of cancer cells, reduction of cancer growth, prolongation of
subject life, palliative effects, or induction of immune response to
cancer cells.

[0044]The term "administering" as used herein refers to the physical
introduction of a composition comprising a cytokine-expressing cellular
vaccine, or a cytokine-expressing cellular vaccine and at least one
additional cancer therapeutic agent or treatment to a patient with
cancer. Any and all methods of introduction are contemplated according to
the invention, the method is not dependent on any particular means of
introduction and is not to be so construed. Means of introduction are
well-known to those skilled in the art, examples of which are provided
herein.

[0045]The term "co-administering" or "co-administered", as used herein
means a process whereby a cytokine-expressing cellular vaccine and at
least one additional cancer therapeutic agent (e.g., AraC) or treatment
to a patient with cancer, are administered to the same patient. The
cytokine-expressing cellular vaccine and AraC are generally administered
sequentially with AraC administered prior to the cytokine-expressing
cellular vaccine. However, the cytokine-expressing cellular vaccine and
AraC may be administered simultaneously or at essentially the same time.
If administration takes place sequentially, the cytokine-expressing
cellular vaccine is typically administered after AraC. The
cytokine-expressing cellular vaccine and AraC may be included in a
therapeutic regimen where an additional cancer therapeutic agent or
treatment is also co-administered. The additional cancer therapeutic
agent or treatment may be administered simultaneously, at essentially the
same time or sequentially to one or both of the cytokine-expressing
cellular vaccine and AraC. The cellular vaccine, AraC and the additional
agent or treatment may be administered one or more times and the number
of administrations of each component of the combination may be the same
or different. In addition, the cytokine-expressing cellular vaccine and
AraC need not be administered at the same site.

[0046]The term "therapeutically effective amount" or "therapeutically
effective combination" as used herein refers to an amount or dose of a
cytokine-expressing cellular vaccine together and the amount or dose of
an additional agent or treatment that is sufficient generate an improved
therapeutic outcome. The amount of cytokine-expressing cellular vaccine
in a given therapeutically effective combination may be different for
different individuals, different tumor types and will be dependent upon
the one or more additional agents or treatments included in the
combination. The "therapeutically effective amount" is determined using
procedures routinely employed by those of skill in the art such that an
"improved therapeutic outcome" results.

[0047]As used herein, the terms "improved therapeutic outcome" and
"enhanced therapeutic efficacy" relative to cancer refers to a slowing or
diminution of the growth of cancer cells or a solid tumor, increased
immune response against the cancer cells, or a reduction in the total
number of cancer cells or total tumor burden. An "improved therapeutic
outcome" or "enhanced therapeutic efficacy" relative to the patient means
there is an improvement in the condition of the patient according to any
clinically acceptable criteria, including an increase in time to tumor
progression, an increase in life expectancy, or an improvement in quality
of life.

[0048]The terms "individual," "subject" as referred to herein is a
vertebrate, preferably a mammal, and typically refers to a human.

[0049]The terms "cancer therapeutic agent," "additional cancer therapeutic
agent or treatment" and the like as used herein refer to any molecule or
treatment that stimulates an anti-cancer response when used alone or in
combination with a cytokine-expressing cellular vaccine (e.g.,
GVAX®). In one aspect, the additional cancer therapeutic agent is
expressed by a recombinant tumor cell and may be an immunomodulatory
molecule, i.e., a second cytokine. In another aspect, the additional
cancer therapeutic agent is administered in the form of a protein or
other chemical entity, e.g., an antibody or standard chemotherapeutic
agent provided in a pharmaceutically acceptable excipient. In yet another
aspect, the cancer therapeutic agent is a standard treatment
traditionally used in the treatment of cancer, e.g., radiation. In a
further aspect, the additional cancer therapeutic agent is an agent or
treatment, which is typically not considered in the treatment of cancer,
but which when administered to a patient in combination with a
cytokine-expressing cellular vaccine results in an improved therapeutic
outcome for the patient under treatment.

General Techniques

[0050]The practice of the present invention will employ, unless otherwise
indicated, conventional techniques of molecular biology, microbiology,
cell biology, biochemistry and immunology, which are within the knowledge
of those of skill of the art. Such techniques are explained fully in the
literature, such as, "Molecular Cloning: A Laboratory Manual," second
edition (Sambrook et al., 1989); "Current Protocols in Molecular Biology"
(F. M. Ausubel et al., eds., 1987); "Animal Cell Culture" (R. I.
Freshney, ed., 1987), each of which is hereby expressly incorporated
herein by reference.

Cancer Targets

[0051]The methods and compositions of the invention provide an improved
therapeutic approach to the treatment of cancer by administration of a
cytokine-expressing cellular vaccine and AraC alone or in combination
with another treatment to a patient with cancer.

[0052]Cancer", "Tumor", or "Neoplasm" as used herein includes cancer
localized in tumors, as well as cancer not localized in tumors, such as,
for instance, cancer cells that expand from a local tumor by invasion
(i.e., metastasis). The invention finds utility in the treatment of any
form of cancer, including, but not limited to, cancer of the bladder,
breast, colon, kidney, liver, lung, ovary, cervix, pancreas, rectum,
prostate, stomach, epidermis; a hematopoietic tumor of lymphoid or
myeloid lineage; acute myeloid leukemia; a tumor of mesenchymal origin
such as a fibrosarcoma or rhabdomyosarcoma; other tumor types such as
melanoma, teratocarcinoma, neuroblastoma, glioma, adenocarcinoma and
non-small lung cell carcinoma.

Introduction Of Cytokine And Cancer Therapeutic Agent Into Cells

[0053]In one aspect of the invention, a nucleic acid chain (i.e., a
recombinant DNA construct or vector) encoding a cytokine operably linked
to a promoter is introduced into a mammalian cell. Any and all methods
for introduction of a recombinant DNA construct or vector into a cell, or
population of cells, typically tumor cells, are contemplated according to
the invention.

[0054]The "vector" may be a DNA molecule such as a plasmid, virus or other
vehicle, which contains one or more heterologous or recombinant DNA
sequences, e.g., a nucleic acid sequence encoding a cytokine under the
control of a functional promoter and in some cases further including an
enhancer that is capable of functioning as a vector, as understood by
those of ordinary skill in the art. An appropriate viral vector includes,
but is not limited to, a retrovirus, a lentivirus, an adenovirus (AV), an
adeno-associated virus (AAV), a simian virus 40 (SV-40), a bovine
papilloma virus, an Epstein-Barr virus, a herpes virus, a vaccinia virus,
a Moloney murine leukemia virus, a Harvey murine sarcoma virus, a murine
mammary tumor virus, and a Rous sarcoma virus. Non-viral vectors are also
included within the scope of the invention.

[0055]Any suitable vector can be employed that is appropriate for
introduction of a recombinant DNA construct into eukaryotic tumor cells,
or more particularly animal tumor cells, such as mammalian, e.g., human,
tumor cells. Preferably the vector is compatible with the tumor cell,
e.g., is capable of facilitating expression of the coding sequence for a
cytokine by the tumor cell, and is stably maintained or relatively stably
maintained in the tumor cell. Desirably the vector comprises an origin of
replication and the vector mayor may not also comprise a "marker" or
"selectable marker" function by which the vector can be identified and
selected. While any selectable marker can be used, selectable markers for
use in such expression vectors are generally known in the art and the
choice of the proper selectable marker will depend on the host cell.
Examples of selectable marker genes which encode proteins that confer
resistance to antibiotics or other toxins include ampicillin,
methotrexate, tetracycline, neomycin (Southern and Berg, J., 1982),
mycophenolic acid (Mulligan and Berg, 1980), puromycin, zeomycin,
hygromycin (Sugden et al., 1985) or G418.

[0056]In practicing the methods of the present invention, a vector
comprising a nucleic acid sequence encoding a cytokine may be transferred
to a cell in vitro, preferably a tumor cell, using any of a number of
methods which include but are not limited to electroporation, membrane
fusion with liposomes, Lipofectamine treatment, high velocity bombardment
with DNA-coated microprojectiles, incubation with calcium phosphate DNA
precipitate, DEAE-dextran mediated transfection, infection with modified
viral nucleic acids, direct microinjection into single cells, etc.
Procedures for the cloning and expression of modified forms of a native
protein using recombinant DNA technology are generally known in the art,
as described in Ausubel, et al., 1992 and Sambrook, et al., 1989,
expressly incorporated by reference, herein.

[0057]Reference to a vector or other DNA sequences as "recombinant" merely
acknowledges the operable linkage of DNA sequences which are not
typically operably linked as isolated from or found in nature. A
"promoter" is a DNA sequence that directs the binding of RNA polymerase
and thereby promotes RNA synthesis. "Enhancers" are cis-acting elements
that stimulate or inhibit transcription of adjacent genes. An enhancer
that inhibits transcription also is termed a "silencer". Enhancers can
function (i.e., be operably linked to a coding sequence) in either
orientation, over distances of up to several kilobase pairs (kb) from the
coding sequence and from a position downstream of a transcribed region.
Regulatory (expression/control) sequences are operatively linked to a
nucleic acid coding sequence when the expression/control sequences
regulate the transcription and, as appropriate, translation of the
nucleic acid sequence. Thus expression/control sequences can include
promoters, enhancers, transcription terminators, a start codon (i.e.,
ATG) in front of the coding sequined, splicing signal for introns and
stop codons.

[0058]Recombinant vectors for the production of cellular vaccines of the
invention provide all the proper transcription, translation and
processing signals (e.g., splicing and polyadenylation signals) such that
the coding sequence for the cytokine is appropriately transcribed and
translated in the tumor cells into which the vector is introduced. The
manipulation of such signals to ensure appropriate expression in host
cells is within the skill of the ordinary skilled artisan. The coding
sequence for the cytokine may be under control of (i.e., operably linked
to) its own native promoter, or a non-native (i.e., heterologous)
promoter, including a constitutive promoter, e.g., the cytomegalovirus
(CMV) immediate early promoter/enhancer, the Rous sarcoma virus long
terminal repeat (RSV-LTR) or the SV-40 promoter.

[0059]Alternately, a tissue-specific promoter (a promoter that is
preferentially activated in a particular type of tissue and results in
expression of a gene product in that tissue) can be used in the vector.
Such promoters include but are not limited to a liver specific promoter
(111 CR, et al., Blood Coagul Fibrinolysis 8 Suppl 2:S23-30, 1997) and
the EF-1 alpha promoter (Kim D W et al. Gene. 91(2):217-23,1990, Guo Z S
et al. Gene Ther. 3(9):802-10, 1996; U.S. Pat. Nos. 5,266,491 and
5,225,348, each of which expressly incorporated by reference herein).
Inducible promoters also find utility in practicing the methods described
herein, such as a promoter containing the tet responsive element (TRE) in
the tet-on or tet-off system as described (ClonTech and BASF), the
metallothienein promoter which can be upregulated by addition of certain
metal salts and rapamycin inducible promoters (Rivera et al., 1996,
Nature Med, 2(9):1028-1032; Ye et al., 2000, Science 283:88-91; Sawyer T
K et al., 2002, Mini Rev Med Chem. 2(5):47588). Large numbers of suitable
tissue-specific or regulatable vectors and promoters for use in
practicing the current invention are known to those of skill in the art
and many are commercially available.

[0060]Exemplary vector systems for use in practicing the invention include
the retroviral MFG vector, described in U.S. Pat. No.5,637,483, expressly
incorporated by reference herein. Other useful retroviral vectors include
pLJ, pEm and [alpha]SGC, described in U.S. Pat. No. 5,637,483 (in
particular Example 12), U.S. Pat. Nos. 6,506,604, 5,955,331-and U.S. Ser.
No. 09/612808, each of which is expressly incorporated by reference
herein.

[0061]Further exemplary vector systems for use in practicing the invention
include second, third and fourth generation lentiviral vectors, U.S. Pat.
Nos. 6,428,953, 5,665,577 and 5,981,276 and WO 00/72686, each of which is
expressly incorporated by reference herein.

[0062]Additional exemplary vector systems for use in practicing the
present invention include adenoviral vectors, described for example in
U.S. Pat. No. 5,872,005 and WO 00/72686, each of which is expressly
incorporated by reference herein.

[0063]Yet another vector system that is preferred in practicing the
methods described herein is a recombinant adeno-associated vector (rAAV)
system, described for example in W098/46728, WO 00/72686, Samulski et
al., Virol. 63:3822-3828 (1989) and U.S. Pat. Nos. 5,436,146, 5,753,500,
6,037,177, 6,040,183 and 6,093,570, each of which is expressly
incorporated by reference herein.

Cytokines

[0064]Cytokines and combinations of cytokines have been shown to play an
important role in the stimulation of the immune system. The term
"cytokine" is understood by those of skill in the art, as referring to
any immunopotentiating protein (including a modified protein such as a
glycoprotein) that enhances or modifies the immune response to a tumor
present in the host. The cytokine typically enhances or modifies the
immune response by activating or enhancing the activity of cells of the
immune system and is not itself immunogenic to the host.

[0065]Exemplary cytokines for use in practicing the invention include but
are not limited to interferons (e.g., IFN-alpha, IFN-beta, and
IFN-gamma), interleukins (e.g., IL-1 to IL-29, in particular, IL-2, IL-7,
IL-12, IL-15 and IL-18), tumor necrosis factors (e.g., TNF-alpha and
TNF-beta), erythropoietin (EPO), MIP3a, macrophage colony stimulating
factor (MCSF), granulocyte colony stimulating factor (G-CSF) and
granulocyte-macrophage colony stimulating factor (GM-CSF). The cytokine
may be from any source, however, optimally the cytokine is of murine or
human origin (a native human or murine cytokine) or is a sequence variant
of such a cytokine, so long as the cytokine has a sequence with
substantial homology to the human form of the cytokine and exhibits a
similar activity on the immune system. It follows that cytokines with
substantial homology to the human forms of IFN-alpha, IFN-beta, and
IFN-gamma, IL-1 to IL-29, TNF-alpha, TNF-beta, EPO, MIP3a, ICAM, M-CSF,
G-CSF and GM-CSF are useful in practicing the invention, so long as the
homologous form exhibits the same or a similar effect on the immune
system. Proteins that are substantially similar to any particular
cytokine, but have relatively minor changes in protein sequence find use
in the present invention. It is well known that small alterations in
protein sequence may not disturb the functional activity of a protein
molecule, and thus proteins can be made that function as cytokines in the
present invention but differ slightly from current known or native
sequences.

Variant Sequences

[0066]Homologues and variants of native human or murine cytokines are
included within the scope of the invention. As used herein, the term
"sequence identity" means nucleic acid or amino acid sequence identity
between two or more aligned sequences and is typically expressed as a
percentage ("%"). The term "% homology" is used interchangeably herein
with the term "% identity" or "% sequence identity" and refers to the
level of nucleic acid or amino acid sequence identity between two or more
aligned sequences, when aligned using a sequence alignment program. For
example, as used herein, 80% homology means the same thing as 80%
sequence identity determined by a defined algorithm, and accordingly a
homologue of a given sequence typically has greater than 80% sequence
identity over a length of the given sequence. Preferred levels of
sequence identity include, but are not limited to, 80, 85, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98 or 99% or more sequence identity to a
native cytokine amino acid or nucleic acid sequence, as described herein.

[0067]Exemplary computer programs that can be used to determine the degree
of identity between two sequences include, but are not limited to, the
suite of BLAST programs, e.g., BLASTN, BLASTX, TBLASTX, BLASTP and
TBLASTN, all of which are publicly available on the Internet. See, also,
Altschul, S. F. et al. Mol. Biol. 215:403410, 1990 and Altschul, S. F. et
al. Nucleic Acids Res. 25:3389-3402, 1997, expressly incorporated by
reference herein. Sequence searches are typically carried out using the
BLASTN program when evaluating a given nucleic acid sequence relative to
nucleic acid sequences in the GenBank DNA Sequences and other public
databases. The BLASTX program is preferred for searching nucleic acid
sequences that have been translated in all reading frames against amino
acid sequences in the GenBank Protein Sequences and other public
databases. In determining sequence identity, both BLASTN and BLASTX
(i.e., version 2.2.5) are run using default parameters of an open gap
penalty of 11.0, and an extended gap penalty of 1.0, and utilize the
BLOSUM-62 matrix. [See, Altschul, et al., 1997, supra.] A preferred
alignment of selected sequences in order to determine "% identity"
between two or more sequences, is performed using for example, the
CLUSTAL-W program in Mac Vector version 6.5, operated with default
parameters, including an open gap penalty of 10.0, an extended gap
penalty of 0.1, and a BLOSUM 30 similarity matrix.

[0068]A nucleotide sequence is considered to be "selectively hybridizable"
to a reference nucleotide sequence if the two sequences specifically
hybridize to one another under moderate to high stringency hybridization
and wash conditions. Hybridization conditions are based on the melting
temperature (Tm) of the nucleic acid binding complex or probe. For
example, "maximum stringency" typically occurs at about TM-5° C.
(5° below the Tm of the probe) "high stringency" at about
5-10° below the Tm; "intermediate stringency" at about
10-20° below the Tm of the probe; and "low stringency" at about
20-25° below the Tm. Functionally, maximum stringency conditions
may be used to identify sequences having strict identity or near-strict
identity with the hybridization probe, while high stringency conditions
are used to identify sequences having about 80% or more sequence identity
with the probe. An example of high stringency conditions includes
hybridization at about 42° C. in 50% formamide, 5×SSC,
5× Denhardt's solution, 0.5% SDS and 100 fig/ml denatured carrier
DNA followed by washing two times in 2×SSC and 0.5% SDS at room
temperature and two additional times in 0.1×SSC and 0.5% SDS at
42° C. Moderate and high stringency hybridization conditions are
well known in the art. See, for example, Sambrook, et al., 1989, Chapters
9 and 11, and in Ausubel, F. M., et al., 1993, (expressly incorporated by
reference herein).

Additional Cancer Therapeutic Agent Or Treatment

[0069]As detailed herein, the present invention is directed to a method of
improving an individual's immune response to cancer (e.g., a target
cancer antigen or antigens) by co-administering a cytokine-expressing
cellular vaccine (e.g., GM-CSF) and AraC for treatment of a patient with
cancer.

[0070]The methods of the invention may comprise the administration of an
additional cancer therapeutic agent other than AraC for use in practicing
the invention. Examples include, but are not limited to, adhesion or
accessory molecules, other biological response modifiers,
chemotherapeutic agents, radiation treatment and combinations thereof.

[0071]Embodiments of the present invention include therapeutic regimens
for treatment of cancer comprising administration of the combination of a
cytokine-expressing cellular vaccine and AraC.

Cellular Vaccine

[0072]Granulocyte-macrophage colony stimulating factor (GM-CSF) is a
cytokine produced by fibroblasts, endothelial cells, T cells and
macrophages. This cytokine has been shown to induce the growth of
hematopoetic cells of granulocyte and macrophage lineages. In addition,
GM-CSF producing tumor cells are able to induce an immune response
against themselves, as well as their parental, non-transduced tumor cell
types.

[0073]Autologous and allogeneic cancer cells that have been genetically
modified to express a cytokine, e.g., GM-CSF, followed by administration
(or in the case of autologous cells, re-administration) to a patient for
the treatment of cancer are described in U.S. Pat. Nos. 5,637,483,
5,904,920 and 6,350,445, expressly incorporated by reference herein. A
form of GM-CSF-expressing genetically modified cancer cells or a
"cytokine-expressing cellular vaccine" for the treatment of pancreatic
cancer is described in U.S. Pat. Nos. 6,033,674 and 5,985,290, expressly
incorporated by reference herein. A universal immunomodulatory
cytokine-expressing bystander cell line is described in U.S. Pat. No.
6,464,973, expressly incorporated by reference herein. Clinical trials
employing GM-CSF-expressing autologous or allogeneic cellular vaccines
have been undertaken for treatment of prostate cancer, melanoma, lung
cancer, pancreatic cancer, renal cancer, and multiple myeloma, and a
number of these trials are currently ongoing.

Combination Therapy: Cytokine-Expressing Cellular Vaccine with AraC

[0074]The present invention provides an improved method of cancer therapy,
which includes slowing the growth of or eradicating pre-existing
malignancies as well as stimulating an immune response to cancer in a
mammalian, preferably a human patient. Desirably, the method effects a
systemic immune response, i.e., a T-cell response and/or a B-cell
response, to the cancer. The method comprises administering to the
patient a cytokine-expressing cellular vaccine and AraC, and may include
another treatment. The cellular vaccine comprises cells which express a
cancer antigen or various cancer antigens, the cancer antigen/antigens
can be one of the antigens of the cancer found in the patient under
treatment. The cells of the vaccine are rendered proliferation
incompetent, for example by irradiation. Upon treatment, the cancer is
eradicated, or its growth slowed, or enters remission, and an immune
response against the cancer is elicited or enhanced. In one approach, the
cytokine-expressing cellular vaccine combination comprises a single
population of cells that is modified to express a cytokine which is
co-administered with at least AraC. In another approach, the vaccine
comprises two or more populations of cells individually modified to
express one component of the vaccine, which are co-administered with
AraC. In yet another approach, the cytokine-expressing cellular vaccine
combination comprises a population of cells that is modified to express a
cytokine which is administered with at least AraC. All of the above
approaches, could also include the co-administration of additional
treatments or therapeutic agents.

[0076]In some embodiments, the cells of the cytokine-expressing cellular
vaccine are cryo-preserved prior to administration. In one aspect of the
invention, the cells of the cytokine-expressing cellular vaccine are
administered to the same individual from whom they were originally
derived (autologous). In another aspect of the invention, the cells of
the cytokine-expressing cellular vaccine and the tumor are derived from
different individuals (allogeneic or bystander). In a preferred approach,
the tumor being treated is selected from the group consisting of cancer
of the bladder, breast, colon, kidney, liver, lung, ovary, cervix,
pancreas, rectum, prostate, stomach, epidermis; a hematopoietic tumor of
lymphoid or myeloid lineage; a tumor of mesenchymal origin such as a
fibrosarcoma or rhabdomyosarcoma; other tumor types such as melanoma,
teratocarcinoma, neuroblastoma, glioma, adenocarcinoma and non-small lung
cell carcinoma.

[0077]In one aspect of the invention, the cells of the cytokine-expressing
cellular vaccine comprises gene-modified cells of one type for the
expression of the cytokine which are administered together with AraC. By
way of example, in one approach, the cytokine-expressing cellular vaccine
is provided as an allogeneic or bystander cell line delivered to the
patient by the intradermal or subcutaneous route while AraC is injected
intravenously. In another approach, the cytokine (i.e., GM-CSF) is
expressed by autologous cells.

[0078]In previous studies, a direct comparison of murine tumor cells
transduced with various cytokines demonstrated that GM-CSF-secreting
tumor cells induced the best overall anti-tumor protection. In one
preferred embodiment, the cytokine expressed by the cytokine-expressing
cellular vaccine of the invention is GM-CSF. The preferred coding
sequence for GM-CSF is the genomic sequence described in Huebner K. et
al., Science 230(4731): 1282-5,1985. Alternatively the cDNA form of
GM-CSF finds utility in practicing the invention (Cantrell et al., Proc.
Natl. Acad. Sci., 82, 6250-6254, 1985).

[0079]Prior to administration, the cells of a cytokine-expressing cellular
vaccine of the invention are rendered proliferation incompetent. While a
number of means of rendering cells proliferation incompetent are known,
irradiation is the preferred method. Preferably, the cytokine-expressing
cellular vaccine is irradiated at a dose of from about 50 to about 200
rads/min, even more preferably, from about 120 to about 140 rads/min
prior to administration to the patient. Most importantly, the cells are
irradiated with a total radiation dose sufficient to inhibit growth of
substantially 100% of the cells, from further proliferation. Thus,
desirably the cells are irradiated with a total dose of from about 10,000
to 20,000 rads, optimally, with about 15,000 rads.

Autologous Cellular Vaccine

[0080]The use of autologous cytokine-expressing cells in a vaccine of the
invention provides advantages since each patient's tumor expresses a
unique set of tumor antigens that can differ from those found on
histologically-similar, MHC-matched tumor cells from another patient.
See, e.g., Kawakami et al., J. Immunol., 148,638-643 (1992); Darrow et
al., J. Immunol., 142,3329-3335 (1989); and Horn et al., J. Immunother.,
10, 153-164 (1991).

[0081]In one embodiment, the present invention comprises a method of
treating cancer by carrying out the steps of: (a) obtaining tumor cells
from a mammal, preferably a human, harboring a tumor; (b) modifying the
tumor cells to render them capable of producing a cytokine or an
increased level of a cytokine naturally produced by the cells; (c)
rendering the modified tumor cells proliferation incompetent; and (d)
re-administering the modified tumor cells to the mammal from which the
tumor cells were obtained or to a mammal with the same MHC type as the
mammal from which the tumor cells were obtained. The administered tumor
cells are autologous or MHC-matched to the host. AraC is co-administered
to the mammal, typically prior to readministering modified
cytokine-expressing tumor cells to the host.

[0082]A cancer treatment method of the invention may rely on the
administration of one or more additional cancer therapeutic agents or
treatments in addition to AraC and modified, cytokine-expressing tumor
cells. The one or more additional cancer therapeutic agents may be
expressed by the same autologous tumor cells that express the cytokine or
the one or more additional cancer therapeutic agents may be expressed by
a different autologous tumor cell population or by a different autologous
tumor cell population using the same or a different vector.
Alternatively, the therapeutic regime comprises administration of
cytokine-expressing cells, AraC and one or more additional cancer
therapeutic treatments such as irradiation or administration of a
chemotherapeutic agent.

Allogeneic Cellular Vaccines

[0083]In one preferred aspect, the invention provides a method for
treating cancer by carrying out the steps of: (a) obtaining a tumor cell
line; (b) modifying the tumor cell line to render the cells capable of
producing a cytokine or an increased level of a cytokine naturally
produced by the cells; (c) rendering the modified tumor cell line
proliferation incompetent; and (d) administering the modified tumor cell
line to a mammalian host having at least one tumor that is the same type
of tumor as that from which the tumor cell line was obtained or wherein
the tumor cell line and host tumor express at least one common antigen.
The administered tumor cell line is allogeneic and is not MHC-matched to
the host. Such allogeneic lines provide the advantage that they can be
prepared in advance, characterized, aliquoted in vials containing known
numbers of cytokine-expressing cells and stored such that well
characterize cells are available for administration to the patient.
Methods for the production of gene-modified allogeneic cells are
described for example in WO 00/72686A1, expressly incorporated by
reference herein. AraC is typically administered to the mammal prior to
administering the modified allogeneic cytokine-expressing tumor cells to
the host.

[0084]In one approach to preparing a cytokine-expressing cellular vaccine
comprising gene-modified allogeneic cells, a cytokine-encoding nucleic
acid sequence is introduced into a cell line that is an allogeneic tumor
cell line (i.e., derived from an individual other than the individual
being treated). In another approach, a cytokine-encoding nucleic acid
sequence and the coding sequence for one or more additional cancer
therapeutic agents are introduced into separate (i.e., different)
allogeneic tumor cell lines. The cell or population of cells may be from
a tumor cell line of the same type as the tumor or cancer being treated.
The tumor and/or tumor cell line may be from any form of cancer,
including, but not limited to, carcinoma of the bladder, breast, colon,
kidney, liver, lung, ovary, cervix, pancreas, rectum, prostate, stomach,
epidermis; a hematopoietic tumor of lymphoid or myeloid lineage; a tumor
of mesenchymal origin such as a fibrosarcoma or rhabdomyosarcoma; or
another tumor, including a melanoma, teratocarcinoma, neuroblastoma,
glioma, adenocarcinoma and non-small lung cell carcinoma.

[0085]In one aspect of the invention, the allogeneic tumor cell is
modified by introduction of a vector comprising a nucleic acid sequence
encoding a cytokine, operably linked to a promoter and expression control
sequences necessary for expression thereof. In another aspect, the same
allogeneic tumor cell or a second allogeneic tumor cell is modified by
introduction of a vector comprising a nucleic acid sequence encoding an
additional cancer therapeutic agent or treatment operably linked to a
promoter and expression control sequences necessary for expression
thereof. The nucleic acid sequence encoding the cytokine and additional
cancer therapeutic agent or treatment may be introduced into the same or
a different allogeneic tumor cell using the same or a different vector.
The nucleic acid sequence encoding the cytokine or cancer therapeutic
agent or treatment mayor may not further comprise a selectable marker
sequence operably linked to a promoter.

[0086]Desirably, the allogeneic cell line expresses GM-CSF in a range from
200-1000 ng/106 cells/24 h. Preferably, the universal bystander cell
line expresses at least about 200 ng GM-CSF/106 cells/24 hours.

[0087]In one embodiment of the invention, one or more allogeneic cell
lines are incubated with an autologous cancer antigen, e.g., an
autologous tumor cell (which together comprise an allogeneic cell line
composition), then the allogeneic cell line composition is administered
to the patient. Typically, the cancer antigen is provided by (on) a cell
of the cancer to be treated, i.e., an autologous cancer cell. In such
cases, the composition is rendered proliferation-incompetent by
irradiation, wherein the allogeneic cells and cancer cells are plated in
a tissue culture plate and irradiated at room temperature using a Cs
source, as detailed above. The ratio of allogeneic cells to autologous
cancer cells in a given administration will vary dependent upon the
combination.

[0088]Any suitable route of administration can be used to introduce an
allogeneic cell line composition into the patient, preferably, the
composition is administered subcutaneously or intratumorally.

[0089]The use of allogeneic cell lines in practicing present invention
provides the therapeutic advantage that, through administration of a
cytokine-expressing allogeneic cell line and at least AraC to a patient
with cancer, in the presence of an autologous cancer antigen, paracrine
production of an immunomodulatory cytokine results in an effective immune
response to a tumor. This obviates the need to culture and transduce
autologous tumor cells for each patient, eliminating the problem of
variable and inefficient transduction efficiencies.

Bystander Cells in Cellular Vaccines

[0090]In one further aspect, the present invention provides a therapeutic
treatment regimen which includes administration of AraC in combination
with a universal bystander cell line that has been transduced to express
an immunomodulatory cytokine. In some cases, the universal bystander cell
line may express both a cytokine and one or more additional cancer
therapeutic agents or each may be expressed by a different universal
bystander cell line. The universal bystander cell line comprises cells
which either naturally lack major histocompatibility class I (MHC-I)
antigens and major histocompatibility class II (MHC-II) antigens or have
been modified so that they lack MHC-I antigens and MHC-II antigens. In
one aspect of the invention, a universal bystander cell line is modified
by introduction of a vector comprising a nucleic acid sequence encoding a
cytokine operably linked to a promoter and expression control sequences
necessary for expression thereof. In another aspect, the same universal
bystander cell line or a second universal bystander cell line is modified
by introduction of a vector comprising a nucleic acid sequence encoding
one or more additional cancer therapeutic agents operably linked to a
promoter and expression control sequences necessary for expression
thereof. The nucleic acid sequence encoding the cytokine and additional
cancer therapeutic agent(s) may be introduced into the same or a
different universal bystander cell line using the same or a different
vector.

[0091]In some cases, the bystander approach is combined with the
autologous or allogeneic approach. For example, an autologous, allogeneic
or bystander cell line encoding a cytokine may be co-administered with
AraC and an autologous, allogeneic or bystander cell line encoding one or
more additional cancer therapeutic agents. The nucleic acid sequence
encoding the cytokine or additional cancer therapeutic agent(s) may or
may not further comprise a selectable marker sequence operably linked to
a promoter. Any combination of a cytokine, AraC and one or more
additional cancer therapeutic agents that stimulate an anti-tumor immune
response finds utility in the practice of the present invention. The
universal bystander cell line preferably grows in defined, i.e.,
serum-free, medium, preferably as a suspension.

[0093]In one embodiment of the invention, a universal bystander cell line
is incubated with a cancer antigen, e.g., an autologous tumor cell or an
allegeneic tumor cell line, which together comprise a universal bystander
cell line composition. This universal bystander cell line composition is
then administered to the patient. Any suitable route of administration
can be used to introduce a universal bystander cell line composition into
the patient. Preferably, the composition is administered subcutaneously
or intratumorally.

[0094]Typically, the cancer antigen is provided by (on) a cell of the
cancer to be treated, i.e., an autologous cancer cell. In such cases, the
composition is rendered proliferation-incompetent by irradiation, wherein
the bystander cells and cancer cells are plated in a tissue culture plate
and irradiated at room temperature using a Cs source, as detailed herein.

[0095]The ratio of bystander cells to autologous or allogeneic cancer
cells in a given administration will vary dependent upon the combination.
With respect to GM-CSF-producing bystander cells, the ratio of bystander
cells to autologous cancer cells in a given administration should be such
that at least 36 ng GM-CSF/106 cells/24 hrs is produced. In general,
the therapeutic effect is decreased if the concentration of GM-CSF is
less than this. In addition to the GM-CSF threshold, appropriate ratios
of bystander cells to autologous tumor cells or tumor antigens can be
determined using routine methods in the art. In one embodiment, the ratio
of bystander cells to autologous cancer cells should not be greater than
1:1.

[0096]The use of allogenic cancer cells or bystander cell lines in
practicing the present invention provides the therapeutic advantage that
it obviates the need to culture and transduce autologous tumor cells for
each patient, eliminating the potential problem of variable and
inefficient transduction efficiencies.

AraC

[0097]AraC (1-β-D-arabinofuranosylcytosine) is one of the older
chemotherapy drugs. It is described in U.S. Pat. No. 3,116,282, issued
Dec. 31, 1963. It is a clear, colorless liquid given by the intravenous,
intrathecal, intraperitoneal or subcutaneous route.

[0098]AraC has a CA registry number of 147-94-4. Its CA name is
4-amino-1-beta-D-arabinofuranosyl-2(1H)-pyrimidinone or
1-beta-D-arabinofuranosylcytosine. AraC is also known as beta-cytosine
arabinosid, aracytidine, Alexan, Arabitin, Aracytine, Cytarbel, Cytosar,
or Udicil.

[0099]AraC is most commonly used in treatment of acute myeloid leukemia,
chronic myeloid leukemia, acute lymphoid leukemia and lymphomas.

[0100]AraC is listed as an antineoplastic or antimetabolite, a class of
drugs that interfere with DNA and RNA. AraC's anti-cancer activity is
associated with its ability to be converted to its biologically active
form, AraCTP. However, AraC is only slowly converted to AraCTP in the
liver or in primary liver tumors due to low levels of an enzyme in the
liver that is required for the conversion of AraC to AraCMP, the first
step in the activation pathway of the drug. Higher doses of AraC cannot
be used to overcome this limitation due to bone marrow toxicity resulting
from rapid activation in that tissue.

[0101]The degree and severity of the side effects depend on the amount and
schedule of Ara-C administration. Some of the most common side effects of
AraC treatment include low white blood counts, low platelet count,
anemia, hair loss, soreness of the mouth, difficulty swallowing, and
diarrhea. In the treatment of AML, even after initial AraC treatments
result in a complete remission of the cancer in the patient, the chance
of cancer recurrence is high.

Evaluation Of Combinations In Animal Models C1498-luc Tumor Model

[0102]The C1498-luc tumor model was developed to evaluate the effects of a
GM-CSF-secreting tumor cell vaccine, C1498.GM, in combination with AraC.
C1498 is a murine AML tumor-derived cell line, and its administration is
an often-used model of AML. In order to monitor leukemia progression in
the animals, C1498 cells were first transduced with a lentiviral vector
encoding the luciferase reporter gene to create the C1498-luc subline. To
assess the in vivo progression of systemic disease, 2.5×104 of
C1498-luc cells were injected intravenously via tail vein into C57BL/6
mice, their syngeneic host, and the mice were examined every few days for
the presence of luminescent signal via live imaging (FIG. 1A). C1498-luc
tumor cells were visualized in the lungs minutes post injection. The
tumor cells then dispersed from the lungs to lymph nodes and bones and
the disease progressed aggressively to detectable systemic lesions within
15 days. In FIG. 1B, animals with photon counts post-tumor challenge are
shown. Whole body photon counts per mouse increased from approximately
5×104 one week post inoculation to greater than
5×108 three weeks post inoculation. Animals with photon counts
exceeding 5×108 exhibited clinical symptoms including ascites,
weight loss and paralysis. Necropsy data, from these animals showed tumor
growth in bone marrow, lymph nodes, spleen, ovaries and ascites and
luciferase-positive tumors were readily visualized by imaging the
isolated organs (data not shown). A total photon count of
5×108 from an individual tumor bearing animal was used as the
end point of life expectancy. The survival of untreated mice was
approximately three to four weeks with an MST of 27 days (data not
shown).

[0103]The efficacy of the combination of cytosine arabinoside (AraC) and
GM-CSF secreting cells was evaluated by carrying out animal studies in
the syngeneic C1498-luc tumor model. In this model, following challenge
with C1498-luc tumor cells as described above, the mice were randomized
into control and individual treatment groups, as detailed in the
examples. For anti-tumor memory assessment, animals which had previously
received AraC in combination with GM-CSF-secreting cells were
rechallenged with a lethal dose of C1498-luc cells approximately 100 days
after receiving the combination therapy. Tumor progression was monitored
by Xenogen imaging in vivo, as shown in FIG. 1A. A typical study in the
C1498-luc tumor model makes use of at least 6 and generally 10-15 mice
per group in order to obtain statistically significant results.
Statistical significance is evaluated using the Student's t-test.

[0104]Immunotherapy with inactivated tumor cells engineered to secrete
GM-CSF is known to elicit long-term systemic, tumor-specific immune
responses. For example, after mice were injected with inactivated B16
cells (C57BL/6 mouse melanoma cell line) virally transduced to express
GM-CSF, subsequent injections of wild-type, non-inactivated B16 cells did
not result in tumor formation. In contrast, injection of wild type
inactivated B16 cells (not expressing GM-CSF) did not protect the mice
from subsequent introduction of live B16 cells, showing the importance of
GM-CSF expression in triggering the immune response.

[0106]The combination of a cytokine-expressing cellular vaccine plus AraC
treatment is expected to increase the efficacy of tumor treatment and
subsequent protection. However, the degree of increased efficacy that
could be expected was not clear, as the efficacy depends on several
factors such as the doses of AraC and the cytokine-expressing cellular
vaccine, as well as the inclusion of another treatment (i.e., dose of the
agent or the frequency and strength of radiation) in the therapeutic
treatment regime. The relative timing and route of administration of
relative to the timing of administration of the cytokine-expressing
cellular vaccine could also impact the therapeutic outcome. Another
concern was that the lymphopenia and neutropenia caused by AraC could
potentially interfere with the development of the long-term immune
response through the vaccination.

Immunological Monitoring

[0107]Several tumor associated antigens have been identified which allow
one to monitor tumor as well as antigen specific immune responses. For
example, tumor antigen-specific T cells can be identified by the release
of IFN-gamma following antigenic restimulation in vitro (Hu, H-M. et al.,
Cancer Research, 2002, 62; 3914-3919). Yet another example of new methods
used to identify tumor antigen-specific T cells is the development of
soluble MHC I molecules also known as MHC tetramers (Beckman Coulter,
Immunomics), reported to be loaded with specific peptides shown to be
involved in an anti-tumor immune response. Examples within the C1498
model include, but are not limited to, gp100, Trp2, Trp-1, and
tyrosinase. Similar melanoma-associated antigens have been identified in
humans. Such tools provide information that can then be translated into
the clinical arena.

Assays For Efficacy Of Combination Therapy In Vivo Models

[0108]Tumor burden is assessed at various time points after tumor
challenge. Typically, spleens cells are assessed for CTL activity by in
vitro whole cell stimulation for 5 days. Target cells are labeled with
51Cr and co-incubated with splenic effector CTL and release of
51Cr into the supernatants as an indicator of CTL lysis of target
cells. On day 3 of in vitro stimulated CTL supernatants are tested for
IFN-gamma production by CTL. In brief, wells are coated with coating
antibody specific for IFN-gamma, supernatant is then added to wells, and
IFN-gamma is detected using an IFN-gamma specific detecting antibody.
IFN-gamma can also be detected by flow cytometry, in order to measure
cell-specific IFN-gamma production.

[0109]Another indication of an effective anti-tumor immune response is the
production of effector cytokines such as TNF-alpha, IL-2, and IFN-gamma
upon restimulation in vitro. Cytokine levels were measured in
supernatants from spleen cells or draining lymph node (dLN) cells
restimulated in vitro for 48 hours with irradiated GM-CSF-expressing
cells.

[0110]A further method used to monitor tumor-specific T cell responses is
via intracellular cytokine staining (ICS). ICS can be used to monitor
tumor-specific T-cell responses and to identify very low frequencies of
antigen-specific T-cells. Because ICS is performed on freshly isolated
lymphocytes within 5 hours of removal, unlike the CTL and cytokine
release assays, which often require 2-7 days of in vitro stimulation, it
can be used to estimate the frequency of tumor antigen-specific T-cells
in vivo. This provides a powerful technique to compare the potency of
different tumor vaccine strategies. ICS has been used to monitor T-cell
responses to melanoma-associated antigens such as gp1OO and Trp2
following various melanoma vaccine strategies. Such T-cells can be
identified by the induction of intracellular IFN-gamma expression
following stimulation with a tumor-specific peptide bound to MHC I.

Xenogen Imaging of Tumor Models

[0111]In some studies, the development and spread of tumors is monitored
by employing the Xenogen whole-animal imaging system. A cancer cell that
has been transduced to express a fluorescent protein, such as luciferase,
is transplanted into a subject, then cancer progression is monitored by
recording in vivo luminescence of the tumor bearing mice. In brief,
Balb/c nu/nu mice are injected with 5×104 or 2×105
cells of C1498-luc cells via tail vein on day O. Mice are monitored for
tumor burden when necessary by intra-peritoneal injection of excess
luciferin substrate at 1.5 mg/g mice weight. In a typical analysis,
twenty minutes after substrate injection, mice are anesthesized and
monitored for in vivo luminescence with Xenogen IVIS Imaging System
(Xenogen Inc.) luminescence sensitive CCD camera by dorsal or ventral
position. Data is collected and analyzed by Living Image 2.11 software.

Cytokine-Expressing Cellular Vaccine Combinations

[0112]The present invention is directed to administration of the
combination of a cytokine-expressing cellular vaccine and AraC to a
cancer patient. The combination may be co-administered with an additional
cancer therapeutic agent or treatment. The additional cancer therapeutic
agent or treatment may be a chemotherapeutic agent, an agent that
modulates the immune response to a cancer antigen, radiation, etc.

[0113]In natural immune responses, CD4+ T helper (Th) cells, reactive
with peptide antigens presented by MHC class II molecules on dendritic
cells (DC), can drive the maturation of DC which is required for
induction of CD8+ CTL immunity. Proper induction, expansion and
maintenance of CTL responses are achieved through the interaction between
CD4+ T cells, DC and CD8+ T cells. While the mechanism is not part of the
invention, the cells to a large extent operate through up-regulation of
CD40L, which interacts with DC-expressed CD40 to effect DC maturation.
CD80/CD86 expressed by mature or activated DC can effect CTL induction by
interaction with the CS28 costimulatory receptor on CD8+ T cells. For
maintenance and full expansion of CTL, interaction of the DC expressed
4-1BB ligand with its receptor 4-1BB on CTL is also important. DC
activation may be triggered by e.g., agonistic anti-CD40 antibody or
ligands of Toll-like receptors (TLR) such as LP5 (TLR4 ligand) or
oligodeoxynucleotides containing CpG-motifs (TLR9 ligand).

Cytokine-Expressing Cellular Vaccines Plus AraC

[0114]The results presented herein demonstrate that the combination of
GM-CSF-secreting C57BL/6 tumor cells and AraC in the treatment of
tumor-bearing subject act synergistically, resulting in significantly
improved survival compared to either treatment being used as a
monotherapy, as well as the establishment of long-term protective
anti-tumor immune responses. In order to achieve the maximal synergistic
effect of these two agents in clinical trials, it is essential to
carefully evaluate possible treatment regimens in preclinical studies. In
studies described herein, the efficacy of the combination was evaluated
in preclinical studies following repeated administration of both AraC and
GM-CSF-secreting tumor cell vaccines in vivo in a murine tumor model.
Example 1 details hematological toxicity of AraC. Example 2 details
studies where AraC and a cytokine-expressing cellular vaccine
(GMCSF-secreting C57BL/6 tumor cells) were tested in the C57BL/6 model as
monotherapies and as a combination therapy (FIG. 3). The combination
therapy of AraC and a GM-CSF secreting tumor cell vaccine together was
dramatically more effective in treating tumors than monotherapy regimens
using AraC or the GM-CSF-secreting tumor cell vaccine separately(Table 1,
FIGS. 3, 4, and 5A-5F).

[0115]These results demonstrate that in practicing the present invention
an autologous, allogeneic, or bystander cytokine-expressing cellular
vaccine may be administered to a cancer patient in combination with an
AraC resulting in enhanced therapeutic efficacy and prolonged survival
relative to either monotherapy alone.

[0116]In a preferred aspect of the methods described herein, a
cytokine-expressing cellular vaccine combination is administered to a
cancer patient, wherein the cytokine expressing cellular vaccine
comprises mammalian, preferably human tumor cells, and the cells in the
cytokine-expressing cellular vaccine are rendered proliferation
incompetent, for example, by irradiation.

[0117]The cytokine-expressing cellular vaccine combination may be
administered by any suitable route. Preferably, the composition is
administered subcutaneously or intratumorally. Local or systemic delivery
can be accomplished by administration comprising administration of the
combination into body cavities, by parenteral introduction, comprising
intramuscular, intravenous, intraportal, intrahepatic, peritoneal,
subcutaneous, or intradermal administration. In the event that the tumor
is in the central nervous system, the composition is administered in the
periphery to prime naive T-cells in the draining lymph nodes. The
activated tumor-specific T-cells are able to cross the blood/brain
barrier to find their targets within the central nervous system.

[0118]In one exemplary embodiment, the cytokine-expressing cellular
vaccine is GM-CSF-expressing cellular vaccine, where the cytokine
expressed is GM-CSF.

[0119]As will be understood by those of skill in the art, the optimal
treatment regimen will vary. As a result, it will be understood that the
status of the cancer patient and the general health of the patient prior
to, during, and following administration of a cytokine-expressing
cellular vaccine combination, the patient will be evaluated in order to
determine if the dose of each component and relative timing of
administration should be optimized to enhance efficacy or additional
cycles of administration are indicated. Such evaluation is typically
carried out using tests employed by those of skill in the art to evaluate
traditional cancer chemotherapy, as further described below in the
section entitled "Monitoring Treatment."

Monitoring Treatment

[0120]One skilled in the art is aware of means to monitor the therapeutic
outcome and/or the systemic immune response upon administering a
combination treatment of the present invention. In particular, the
therapeutic outcome can be assessed by monitoring attenuation of tumor
growth and/or tumor regression and or the level of tumor specific
markers. The attenuation of tumor growth or tumor regression in response
to treatment can be monitored using several end-points known to those
skilled in the art including, for instance, number of tumors, tumor mass
or size, or reduction/prevention of metastasis.

[0122]Cell Lines and Reagents. C1498, a murine AML cell line, was
purchased from American Type Culture Collection (ATCC, Manassas, Va.).
C1498 was originally derived from a female C57Bl/6J (H-2b) mouse and
subsequently adapted to tissue culture and is MHC class I.sup.+ and MHC
class II.sup.+. The C1498-luc subline was established by transduction of
C1498 with lentiviral vector expressing a luciferase reporter gene, and
the C1498.GM subline by transduction of C1498 with lentiviral vector
expressing mouse GM-CSF. The latter generates 70 ng of mouse GM-CSF per
106 cells per 24 hours in culture. Both transduced cell lines were
maintained in culture conditions recommended by ATCC. Cytarabine, also
known as cytosine arabinoside or AraC, was purchased from Cardinal
Health, San Diego, Calif.

[0123]Mice. Female C57Bl/6 mice and female C57Bl/6 congenic Thy 1.1 mice
were purchased from Taconic (Oxnard, Calif.) and the Jackson Laboratory
(Bar Harbor, Me.) respectively, and maintained according to institutional
and NIH guidelines. All mice were used between 8 and 12 weeks of age.
Water and food were provided ad libitum.

[0124]Tumor Model. Female C57Bl/6 mice were challenged with C1498-luc
cells via intra-tail-vein injections with 2.5×104 inocula. The
mice were prepared for in vivo bioluminescence imaging 5 to 10 minutes
post injection to confirm the initial trafficking of the tumor cells from
tail vein to the lungs. Briefly, the mice were injected i.p. with 1.5
mg/g luciferin substrate (Xenogen Corp., Alameda, Calif.). Fifteen
minutes later, the mice were anesthetized for in vivo bioluminescence
imaging analysis. Nearly 100% of challenged mice imaged positively,
demonstrating initial trafficking of C1498-luc to the lungs. The animals
were monitored by in vivo imaging every 5 to 7 days throughout the study
to monitor the systemic progression of the tumor. Individual animals were
euthanized when in vivo total photon counts exceeded 5×108
and/or when determined to be clinically paralyzed.

[0125]Hematologic and Phenotypic Analysis. Mice were injected
intraperitoneally with AraC using the treatment regimen described below.
Peripheral blood was collected by retro-orbital puncture into EDTA-coated
capillary tubes on days 1, 2, 3, 4, 6, 8 and 11. Hematologic analysis was
performed by IDEXX pre-clinical Research Services (West Sacramento,
Calif.).

[0126]In Vivo Treatment. Following challenge with C1498-luc tumor cells as
described above, the mice were randomized into control and individual
treatment groups. For AraC treatment; at 24 hours post challenge, the
animals received three i.p. injections of 100 mg/kg AraC (volume of
injection: 200 μl) in 10 hour-increments. This treatment regimen is
equal to a total dose of 900 mg/m2 (300 mg/m2 per injection)
and is within the total dose range of 700 to 1400 mg/m2 used
clinically in human patients. This dose level is typical for inducing
remission, which is a dosage substantially lower than a typical
post-remission high-dose consolidation regimen (e.g., 10 doses of 3000
mg/m2 for a total dose of 30,000 mg/m2). For C1498.GM
treatment, 7 days post challenge; the animals were given a single dose of
irradiated C1498.GM cells at 1×106/500 Jμl subcutaneously.
For the combination therapies, 3 AraC injections at 10-hour intervals
were given on day 2 followed by a single C1498.GM injection 3, 5, or 7
days post AraC, at the nadir, rebound or at the recovered phase of
lymphopenia and neutropenia induced by AraC, respectively. For long-term
anti-tumor memory assessment, animals which had previously received AraC
in combination with GM-CSF-secreting Tumor Cell Immunotherapy were
rechallenged with a lethal dose of 5×104 C1498-luc cells
approximately 100 days after receiving the combination therapy. A group
of five naive mice were received the same inoculum of C1498.luc as
control. Tumor progression was monitored by Xenogen imaging in vivo.

[0128]51Cr Release Cytotoxicity Assay. Activity of cytotoxic T-Iymphocytes
(CTL) was assessed using the standard 51Chromium-release assay.
Briefly, 2×106 target cells were labeled at 37° C. for
1 h with 100 μCi Na251CrO4 (MP Biomedicals). Target cells
were washed 3× and resuspended to 5×104 cells/ml. Five
thousand radiolabeled target cells per well (100 μl) were added to a
96 well plate, together with the appropriate number of effector cells
(100 μl/well). The defined effector:target (E:T) ratios were plated in
triplicate. Cytotoxicity assays were performed at 37° C. for 4 h.
After incubation, cell-free supernatants were collected and analyzed in a
gamma counter. Percent specific lysis was calculated using the following
equation: (ER-SR)/(MR-SR)×100, where ER=experimental release,
SR=spontaneous release and MR=maximum release.

EXAMPLE 1

Characterizing Hematological Toxicity of AraC Treatment

[0129]Prior to conducting the in vivo efficacy studies in the leukemia
tumor models, the hematological toxicity of AraC was determined. The
animals received three i.p. injections of 100 mg/kg AraC (volume of
injection: 200 μl) in 10 hour-increments. This treatment regimen is
equal to a total dose of 900 mg/m2 and is within the total dose
range of 700 to 1400 mg/m2 used clinically in human patients.
Neutropenia and lymphopenia are known to be the primary dose-limiting
toxicity observed in patients, so AraC treated mice were monitored for
absolute neutrophil and lymphocyte counts. The results are shown in FIG.
2A and FIG. 2B, respectively. After the three AraC administrations,
peripheral blood was collected by retro-orbital puncture into EDTA-coated
capillary tubes on days 1, 2, 3, 4, 6, 8 and 11 and analyzed for
neutropenia. The absolute neutrophil count in the animals dropped to 45
neutrophils/μl, by day 4, rebounded to control level at 600
neutrophils/μl, by day 6 and remained at a steady level of around 600
neutrophils/μl, thereafter (FIG. 2A), which is similar to untreated
control animals. Similar effects were observed on lymphocyte counts,
which dropped by greater than 40% by day 3 and rebounded by day 6 (FIG.
2B).

EXAMPLE 2

Combination Therapy: Cytokine-Expressing Cellular Vaccine and AraC

[0130]In vivo studies were carried out using the C57BL/6 model to
determine if AraC in combination with a cytokine-expressing cellular
vaccine can enhance anti-cancer efficacy. The optimal timing of AraC
administration relative to GM-CSF-expressing cellular vaccine was also
investigated.

[0131]C57BL/6 mice were inoculated intravenously via tail vein on day 0
with 2.5×104 C1498-luc cells expressing the luciferase
reporter gene. The C1498-luc tumor bearing mice were treated with either
AraC or C1498.GM or in combination. For monotherapy, AraC was
administered one day post tumor challenge by three intraperitoneal
injections at 100 mg/kg 10 hours apart or 1×106 irradiated
C1498.GM cells were administered as a single subcutaneous injection to
designated animals on day 7 post tumor challenge. The animals receiving
combination therapies were given the two agents sequentially, scheduled
as for monotherapy. The dose levels of AraC and C1498.GM given in this
experiment did not result in substantial anti-tumor activity in the
C1498-luc tumor model when used as monotherapy, however, detection of
synergistic effects of the two therapies were readily detectable (FIG.
3). At the dose levels used in this experiment, both AraC and C1498.GM as
monotherapies had modest positive effects on survival of C1498-luc tumor
bearing animals, with only 30% of animals surviving the disease at 150
days post-challenge. The combination of AraC and C1498.GM significantly
prolonged survival of tumor bearing mice over either monotherapy
regimens. The majority (90%) of mice receiving the combined therapy
survived for longer than 150 days, while only 30% of mice receiving
either AraC or c1498.GM monotherapy survived for over 150 days. In
addition, the surviving mice of the combination therapy group were tumor
free at the 150 day time point, while none of the monotherapy mice were,
as shown in Table 1.

[0132]Mice surviving from the combination therapy group (n=10) then
underwent tumor challenge again, with 5×104 live C1498-luc
cells, a lethal dose, administered on day 143 after receiving the
combination therapy. Upon rechallenge, none of the animals that
previously received combination therapy developed tumors, and they
remained tumor-free for the duration of the study (>100 days),
demonstrating the existence of a long term protective response against
the cancer cells (FIG. 4). The presence of this long-term protection
would also prevent a post-remission recurrence of the cancer.

[0133]Tumor-specificity of T-cell responses were evaluated in animals
treated with C1498.GM monotherapy or AraC plus C1498.GM combination
therapy. This set of experiments was carried out in Thy 1.1 congenic mice
to permit depletion of the tumor cells, which were Thy 1.2.sup.+. On day
0, Thy 1.1 congenic C57BL/6 mice were intravenously challenged with
2.5×104 live Thy 1.2 C1498-luc leukemia cells. Starting on day
1, three i.p. injections of AraC were administered 10 hours apart to mice
designated to receive the combination therapy. On day 7, 1×106
irradiated C1498.GM cells were given as monotherapy alone or to AraC
treated mice that received the combination therapy. On day 21, spleen
cells from the mice (n=5 per group) were harvested and depleted of the
Thy 1.2.sup.+ C1498-luc tumor cells using anti-Thy1.2 MACs beads.
Tumor-depleted splenocytes were confirmed by flow cytometry to contain
less than 1% Thy 1.2.sup.+ C1498-luc cell. Splenocytes were co-cultured
with irradiated C1498 cells as stimulators at a 25:1 ratio for five days
with five units/mL of murine IL-2 added to the culture on day 2.
51Cr labeled CTLL2 (a syngenetic lymphoblast cell line) or C1498
cells were used as control and target cells, respectively in the 4 hour
51Cr release assay. Splenocytes from C1498.GM monotherapy and AraC
plus C1498.GM treated mice did not exhibit any cytolytic activity against
the CTLL2 control cells at the effector to target ratios evaluated. In
contrast, splenocytes from C1498.GM monotherapy or AraC plus C1498.GM
treated mice demonstrated comparable cytolytic activity against C1498
target cells which, as expected, was dependent on the effector to target
ratio (FIGS. 5B and 5C). Splenocytes from HBSS injected control animals
did not demonstrate any cytolytic activity against either of the two
target cells. Furthermore, evaluating the tumor-depleted splenocytes for
activation markers revealed similar phenotypic patterns for mice treated
with C1498.GM monotherapy or AraC plus C1498.GM. Both groups of mice
showed a significant increase in the percentage of CD8 cells expressing
CD107a.sup.+ (a marker for CD8 cell cytolytic activity; FIG. 5D),
CD44hiCD62L1o (markers for CD8 cell migration and homing; FIG.
5E), and NKG2D.sup.+ (a CD8 cell activation marker; FIG. 5F) compared to
HBSS treated control mice, indicating that, similarly to previous
examples of cell-based cancer vaccines employing GM-CSF, the development
of the specific immune response was correlated with the activation of CD8
cytotoxic activity. Moreover, the percentage differences in activation
markers in the CD8 subpopulation of mice treated with C1498.GM
monotherapy compared to AraC plus C1498.GM combination therapy were not
significant. Taken together, data from immune monitoring assays suggests
that the co-administration of AraC does not interfere with immunotherapy
using GM-CSF-secreting cancer cells, and that the combination therapy was
successful in inducing specific anti-cancer immune response despite the
AraC-induced lymphopeia and neutropenia.

[0134]A further indication as to the utility of combining a GM-CSF
secreting cellular vaccine and AraC in eliciting an anti-tumor immune
response is the production of effector cytokines such as TNF-alpha, IL-2,
and IFN-gamma upon restimulation in vitro. Release of such cytokines is
often used as a surrogate marker for monitoring tumor specific immune
responses following immunotherapeutic strategies designed to induce
anti-tumor immunity. Cytokine levels were measured in supernatants from
spleen cells restimulated in vitro for 48 hours with irradiated
GM-CSF-secreting tumor cells. The presence of the GM-CSF-secreting tumor
cells induced the production of TNF-alpha, IFN-gamma, IL-5 and IL-2 by
the spleen cells.